CGTase (EC 2.4.1.19) is an enzyme functioning on α-1,4-glucans such as starches to produce cyclodextrins (CD), which are cyclic α-1,4-glucans, through its intramolecular transfer activity. The degree of polymerization of the CDs produced by CGTase is chiefly 6-8, with these products being referred to as α-, β and γ-CD, respectively. In addition to this CD-producing reaction, CGTase catalyzes coupling reactions (the ring of the CD is opened and the resulting straight-chain oligosaccharide is transferred to a receptor sugar molecule) and disproportionation reactions (a straight-chain oligosaccharide is transferred to the receptor sugar molecule) through the intramolecular transfer reaction. Further, albeit weakly, CGTase also catalyzes the hydrolysis reaction of the α-1,4-glucoside bond.
Since CDs can change chemical and physical properties of various molecules by making clathrates therewith, CGTase has achieved a position as an important enzyme in the food, pharmaceutical and cosmetic industries. Thus, beginning from the CD synthesis reaction by the Bacillus macerans enzyme in 1939 (E. B. Tilden and S. J. Pirt, J. Am. Chem. Soc., 63, 2900-2902, 1939), a large number of studies were conducted, including a search for bacteria producing CGTase and means of purifying the enzyme (Sumio Kitahata, Naoto Tsuyama and Shigetaka Okada, Agr. Biol. Chem., 38 (2), 387-393, 1974; Sumio Kitahata and Shigetaka Okada, Agr. Biol. Chem., 38 (12), 2413-2417, 1974; Sumio Kitahata and Shigetaka Okada, J. Jap. Soc. Starch Sci., 29 (1), 13-18, 1982; Michio Kubota, Yoshiki Matsuura, Shuzo Sakai and Yukiteru Katsube, Denpun Kagaku, 38 (2), 141-146, 1991; Lionel J. Bovetto, Daniel P. Backer, Jaques R. Villette, Philippe J. Sicard, and Stephane J-L. Bouquelet, Biotechnology and Applied Biochemstry, 15, 48-58, 1992; Shinske Fujiwara, Hirofumi Kakihara, Kim Myung Woo, Andre Lejeune, Mitsuhide Kanemoto, Keiji Sakaguchi, and Tadayuki Imanaka, Applied and environmental microbiology, 58 (12), 4016-4025, 1992; Florian Binder, Otto Huber and August Bock, Gene, 47, 269-277, 1986; Keiji Kainuma, Toshiya Takano and Kunio Yamane, Appl. Microbiol. Biotechnol., 26, 149-153, 1987; Takahiro Kaneko, Tetsuo Hamamoto and Koki Horikoshi, J. general Microbiology, 134, 97-105, 1988; Murai Makela, Pekka Mattsson, M. Eugenia Schinina, and Timo Korpela, Biotechnology and Applied biochemistry, 10, 414-427, 1988; Ernest K. C. Yu, Hiroyuki Aoki, and Masanaru Misawa, Appl. Microbiol. Biotechnol., 28, 377-379, 1988).
Based on the type of CD principally synthesized, CGTase is classified as α-CGTase, β-CGTase or γ-CGTase. Most of what has been reported in the past has related to α-, or β-CGTase. Few enzymes have been reported as being γ-CGTase (Shigeharu Mori, Susumu Hirose, Takaichi Oya, and Sumio Kitahata, Oyo Toshitsu Kagaku, 41 (2), 245-253, 1994; Yoshito Fujita, Hitoshi Tsubouchi, Yukio Inagi, Keiji Tomita, Akira Ozaki, and Kazuhiro Nakanishi, J. Fermentation and Bioengineering, 70 (3), 150-154, 1990; and Takashi Kato and Koki Horikoshi, J. Jpn. Soc. Starch Sci., 33 (2), 137-143, 1986).
Enzymes reported to be γ-CGTase is not industrially available because the quantity of γ-CD produced is not more than 5 percent, the rate of production of β-CD accelerates in the later stages of the reaction and thus the amount of β-CD produced is equal to or greater than the amount of γ-CD produced, or the amount of γ-CD produced drops precipitously at a substrate concentration of 10 percent or greater and, as a countermeasure, ethanol must be present together in the reaction solution.
On the other hand, attempts have also been made to modify the structural genes of α- or β-CGTase to improve the quantity of γ-CD produced (Akira Nakamura, Keiko Haga, and Kunio Yamane, Biochemstry, 32, 6624-6631, 1993; and Michio Kubota, Yoshiki Matsuura, Shuzo Sakai and Yukiteru Kutsume, Oyo Toshitsu Kagaku, 41 (2), 245-253, 1994). However, these are also inadequate from an industrial perspective, because even when the quantity of γ-CD produced is increased, the β-CD produced by the original activity is not decreased substantially.
Thus, although α-CD and β-CD are employed in various fields, γ-CD is currently little employed. The same is true for CD-containing syrups. CD syrups comprising principal components in the form of α-CD or β-CD are employed in various fields, while CD syrup comprising γ-CD as principal component are seldom employed.